The present invention relates to molecular reagents and molecular diagnostic tests useful for detecting certain proteins produced by tumors.
Proteins that are specifically associated with certain tumors have been described as early as 1965. These tumor associated proteins or tumor markers would be a powerful tool in detecting certain malignancies if they were qualitative. Most of the currently identified tumor markers are quantitative instead of qualitative. Garrett and Kurtz (Medical Clinics of North America, 70:1295-1306 (1986)) defined the ideal biological tumor marker as one that is a) specific without false-positive results, b) sensitive without false-negative results and c) capable of demonstrating an absolute correlation with the extent of disease. To date, for a variety of reasons, unique and specific tumor markers have not been adequately characterized. The broad classification of tumor markers can be divided into two groups: (1) tumor associated proteins and (2) oncogene products.
A wide variety of tumor associated proteins found in certain tumor tissues and serum have been described. They include carcinoembryonic antigen (CEA), tumor associated glycoprotein (TAG), CA19-9, human chorionic gonadotropin (HCG), alpha-feto protein (AFP), and a 145,000 nucleolar protein (p145) (Schlom, J. Cancer Research 46:3225-3238 (1986); Chan, J. C., Cancer Bulletin 40:213-217 (1988); Freeman, J. W., et al, Cancer Research 46:3593-3598 (1986); Gion, M., et al, International Journal of Biological Markers, 1:33-38 (1986)). All of these proteins are associated with a wide range of human malignancies.
Oncogene products are a second category of tumor markers found in human tumors. Proto-oncogenes are normal cellular genes which are involved in normal growth and differentiation. These proto-oncogenes can transform into oncogenes by one of the following mechanisms: point mutations in the coding region, amplification of genes, or chromosomal translocation. Once a proto-oncogene is changed into an oncogene, neoplastic growth occurs. The ras and myc families of cellular oncogenes are two of the most frequently identified activated oncogenes. The ras family of oncogenes consist of the Harvey ras, Kirsten ras and neuroblastoma ras oncogene. Activation of ras oncogene causes an increase of ras specific protein (p21) in colon, colorectal, lung, mammary, neuroblastoma, prostate, ovarian, melanoma and bladder carcinomas (Der, C. J. and Cooper, G. M., Cell 32:201-208 (1983); Feig, L. A., et al, Science 223:698-700 (1984); McCoy, M. S., et al, Nature 302:79-81 (1983); Viola, M. V., et al. New England Journal of Medicine 314:133-137 (1986). Thus, one can readily deduce that the p21 ras oncogene protein is a powerful tumor marker.
In higher eukaryotic cells, the regulation of gene expression is often mediated by turning on and off RNA synthesis in a temporally ordered manner. A popular hypothesis for the synthesis of messenger RNA by RNA polymerase II, involves specific promoter interaction with one or more cellular transcriptional factors. To be effective, these transcriptional factors must recognize and bind to specific DNA sequences located within the promoter region of the eukaryotic gene of interest. Most researchers believe that both temporal regulation and tissue specificity of transcription are controlled by these cellular DNA binding proteins (Dynan, W. S. and Tijian, R., Cell 35:79-87 (1983)). For accurate and optimal initiation of transcription, the structural organization of different promoters recognized by polymerase II involve multiple common elements located upstream of the RNA start site. Specific DNA sequences for the promoter region of a gene (consensus sequences) are recognized by a limited number of nuclear proteins. A variety of nuclear proteins (SPI, NFI, API and COUP) have been shown to specifically recognize and interact with certain promoter sequences.
Since oncogene products belong to a broad classification of tumor markers, the identification of a tumor marker protein which specifically bound to a region of the human Harvey ras promoter would be very useful and powerful. Any tumor markers identified in this manner may be transcriptional activators of the Harvey ras oncogene. Since the p21 Harvey ras protein product is found in a variety of human malignancies (colon, colorectal, lung, mammary, neuroblastoma, prostate, ovarian, melanoma and bladder), identifying a transcriptional activator of the Harvey ras oncogene would be most beneficial.
There remains a profound need for an improved method of identifying human malignancies at an early stage. The present invention provides a reliable, sensitive, specific, efficient and reproducible method for the detection of circulating serum tumor markers (factors) specific to the ras oncogene promoter.